Thomas Hohn

Expression from polycistronic cauliflower mosaic virus pregenomic RNA

Cauliflower mosaic virus (CaMV), a plant pararetrovirus, shares many properties with true retroviruses, such as genome replication by transcription/reverse transcription and organisation of the GAG- and POL-genes (Fig.1). It also has a transactivator gene (TAV; Bonneville at al., 1989; Gowda et al., 1989), like the lentiviruses. Plant viruses spread through their host via plasmodesmata, narrow cytoplasmic connections between cells, and are transmitted by insects, while mammalian retroviruses leave and re-enter cells with the help of envelopes. Reflecting these different mechanisms of virus spread in animals and plants, the CaMV genome includes genes for systemic spreading (SYS) and for insects transmissibility (ITS; Fig. 1) but misses an envelope gene.

Evaluation of Constitutive Cestrum Yellow Leaf Curling Virus Promoter in Maize and Tomato

We have cloned and evaluated two versions of a novel, strong and constitutive promoter from Cestrum yellow leaf-curling virus (CmYLCV) called CmpC (short- 346bp) and CmpS (longer- 400bp), which can be used for regulating transgene expression in a wide variety of plant species. CmYLCV belongs to the Caulimoviridae family and was first reported in Cestrum parqui from the Solanaceae by Ragozzino (1974). Recently, CmYLCV was cloned and seven open reading frames were identified in the genomic sequence (Hohn et al., 2001). To evaluate the utility of the CmYLCV promoter to drive expression of heterologous genes in plants, two versions of the full-length transcript promoter were cloned in front of the GUS, CAT and FP reporter genes and tested in transient assays in Nicotiana plumbaginifolia, Orichophragmus violaceus and Oriza sativa protoplasts as well as in stably transformed Zea mays and Lycopersicon esculentum. The transient expression experiments show that, depending on the plant system used, the expression level of CmpC and CmpS promoter fragments are higher than the expression level of the widely used 35S promoter from Cauliflower Mosaic Virus (Hohn et al., 2001) and that the longer promoter fragment is the weaker one. Expression analysis of CmpC and CmpS promoter fragments in stably transformed maize (Figurel) have shown that both fragments are on average ten times greater than the strong constitutive Ubi 1 promoter from Z. mays (Christensen et al., 1992) and that the expression levels in tomato (Figure 2) are comparable with the strong, constitutive SMAS promoter (Ni et al., 1994). Moreover, the spatial expression analysis has shown that both promoters express in various tissue types, except pollen in both maize and tomato and that both promoter fragments retain their high expression levels through at least two generations. We limited our analysis to the single copy events only (Ingham et al., 2001) since it is a widely accepted idea that the copy number of a transgene affects the expression level in transgenic plants.

Polyadenylation of Cauliflower Mosaic Virus RNA is Controlled by Promoter Proximity

Sequences required for efficient cauliflower mosaic virus RNA polyadenylation and dependence of polyadenylation on promoter distance were determined. A comparison of polyadenylation mechanisms for two retro- and two pararetroviruses is given.